Fusion proteins having mutated immunoglobulin hinge region

ABSTRACT

A fusion protein having a non-immunoglobulin polypeptide having a cysteine residue proximal to the C terminal thereof, and an immunoglobulin component with a mutated hinge region is provided. The mutation comprises a point mutated site corresponding in position to the position in a native hinge region of the cysteine residue located nearest the cysteine residue of the non-Ig component. The distance from the cysteine residue of the non-immunoglobulin polypeptide and any remaining cysteine residues of the mutated hinge region is sufficient to prevent the formation of a disulphide bond therebetween.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of pending U.S. patent application Ser.No. 12/180,455 filed 25 Jul. 2008 which claims the benefit of U.S.provisional patent application No. 60/952,181 filed 26 Jul. 2007. Thesubject matter of this application is related to U.S. patent applicationSer. No. 11/340,661 filed 27 Jan. 2006, now U.S. Pat. No. 7,625,564,issued 1 Dec. 2009, and PCT patent application No. PCT/CA2007/000107filed 25 Jan. 2007, published as US 2009/0297522 on 3 Dec. 2009. Thedisclosure of each of the previously referenced patent application(s)and patent(s) is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

This application relates to fusion proteins.

BACKGROUND

Recombinant human proteins corresponding to their natural amino acidsequences have been used for the treatment and diagnosis of a broadrange of human diseases since the 1980s. However, most recombinant humanproteins do not survive long enough in vivo and are rapidly cleared fromcirculation. For example, proteins with a molecular mass less than 20kDa have been reported to be filtered at the level of renal tubules,often leading to a dose-dependent nephrotoxicity. The short in vivohalf-life of these proteins compromises their natural biologicalfunctions, requiring higher doses or more frequent administration, whichin turn impairs patient compliance and increases the burden on healthcare providers. These clinical demands merit the search and developmentof therapeutic proteins with longer circulation half-life.

In addition to the direct mutations of individual protein structure forachieving longer half-life (e.g. ARANESP™ by Amgen and TNKnase byGenentech), two systemic approaches have been used for the creation oftherapeutic proteins with longer half-life. One is “PEGylation”, whichrefers to chemical cross-linking of polyethylene glycol (PEG) compoundsto target proteins. PEG-bound proteins have larger molecular sizes andare more slowly cleared from the circulation. PEGylation has beenclinically demonstrated and recognized by the biotech industry as astandard method of extending the half-life of various target proteins. Ashortcoming of PEGylation is the significant impairment of thebiological activity of target proteins. The altered structure ofPEGylated proteins also risks generating an immunogenic response in thehuman body.

Another systemic approach is the genetic fusion of target therapeuticprotein(s) with another human carrier protein to stabilize the targetprotein in circulation in the form of a fusion protein complex. Twoideal human carrier protein candidates for fusion with therapeuticproteins are human immunoglobulin and albumin. Both immunoglobulin andalbumin are very stable and abundant in blood. Fusion proteinscomprising a therapeutic protein and either immunoglobulin or albuminwould theoretically retain the biological activity of the therapeuticprotein, be more stable in circulation than the therapeutic proteinalone, and be completely homologous to natural human proteins,minimizing the risk of immunogenic responses [1, 2].

One practical strategy with this approach is to genetically fuse atherapeutic protein with an Fc fragment of a human immunoglobulin [1, 3,4]. Modern bioengineering technology has successfully created fusionproteins consisting of a therapeutic protein, such as cytokines andsoluble receptors, and an Fc fragment of immunoglobulin G (IgG) [5-26].For example, IL-10, an anti-inflammatory and anti-rejection agent, hasbeen fused to the N-terminal of murine Fc.gamma.2a to increase IL-10'sshort circulating half-life [9]. In another example, the N-terminal ofhuman IL-2 has been fused to the Fc portion of human IgG 1 or IgG 3 toovercome the short half life of IL-2 and its systemic toxicity [26]. Twofusion proteins comprising an Fc fragment have been successfullydeveloped as biomedicines and approved by FDA for the treatment ofrheumatoid arthritis and chronic plaque psoriasis [27, 28, 29].

Human IgG is composed of four polypeptides (two identical copies oflight chain and heavy chain) covalently linked by disulfide bonds. Theproteolysis of IgG by papain generates two Fab fragments and one Fcfragment. The Fc fragment consists of two polypeptides linked bydisulfide bonds. Each polypeptide, from the N-terminal to C-terminal, iscomposed of a hinge region, a CH2 domain and a CH3 domain. The structureof the Fc fragment is nearly identical across all subtypes of humanimmunoglobulin. IgG is one of the most abundant proteins in the humanblood and makes up 70 to 75% of the total immunoglobulin in human serum.The half-life of IgG in circulation is the longest among all five typesof immunoglobulin and may reach 21 days.

Disulfide bonds formed between thiol groups of cysteine residues play animportant role in the folding and stability of proteins, usually whenproteins are secreted to an extracellular medium. The disulfide bondstabilizes the folded form of a protein in several ways. First, it holdstwo portions of the protein together, biasing the protein towards thefolded state. Second, the disulfide bond may form the nucleus of ahydrophobic core of the folded protein, i.e., local hydrophobic residuesmay condense around the disulfide bond and onto each other throughhydrophobic interactions. Third, and related to the first and secondpoints, by linking two segments of the protein chain and increasing theeffective local concentration of protein residues, the effective localconcentration of water molecules is lowered. Since water moleculesattack amide-amide hydrogen bonds and break up secondary structures,disulfide bonds stabilize secondary structure in their vicinity. Forexample, researchers have identified several pairs of peptides that areunstructured in isolation, but adopt stable secondary and tertiarystructure upon forming a disulfide bond between them. The native form ofa protein is usually a single disulfide species, although some proteinsmay cycle between a few disulfide states as part of their function. Inproteins with more than two cysteines, non-native disulfide species,which are almost always unfolded, may be formed.

A flexible junction region of the fusion protein which allows the twoends of the molecule to move independently plays a very important rolein retaining each of the two moieties' functions separate and efficient.Therefore, the junction region should act as a linker which combines thetwo parts together, and as a spacer which allows each of the two partsto form its own biological structure and not interfere with the otherpart. Furthermore, in order to avoid the induction of immunogenicity,the junction region should be native to the human body and simple instructure [5, 25].

The primary structure of the hinge region of immunoglobulin includesthree cysteines, such as cys²²³, cys²²⁹ and cys²³² in the case of thehuman IgG 1 structure used by the present inventors. While the cys²²⁹and cys²³² foam two interchain disulfide bonds by binding betweencounterparts of the two chains, the cys²²³ remains free. Therefore, itis highly possible that this free cysteine may bind with anotherintrachain or interchain cysteine, to form a non-native disulfide bondin the protein maturation process upon secretion from host cells orduring subsequent purification. This non-native disulfide bond may notonly alter the structure and conformation of the therapeutic protein,but may also interfere with the biological activity of the therapeuticprotein or induce harmful immunogenicity when the fusion protein isadministrated into the human body.

Many therapeutic proteins such as erythropoietin (EPO) and granulocytemacrophage colony-stimulating factor (GM-CSF) have a cysteine near theirC-terminal. The role of this cysteine in maintaining proper structureand function has yet to be well-defined. The cysteine proximal to theC-terminal may be essential for maintaining proper structure,facilitating correct folding or retaining normal biological activity.The inventors hypothesize that if proteins with a cysteine near itsC-terminal are fused to the natural sequence of the hinge region of a Fcfragment, the very limited space between the last cysteine of theC-terminal of the fused protein and the first cysteine of the N-terminalof the Fc fragment (cys²²³) may lead to the formation of an unexpecteddisulfide bond between these two cysteines. The formation of theunexpected disulfide bond may alter the structure and/or the folding ofthe fused protein component as well as alter the flexibility of thehinge region. As a result, normal functions of the fused therapeuticprotein in the fusion protein complex may be impaired.

Even if the target therapeutic protein does not contain a cysteine nearits C-terminal, another cysteine in its structure may, after threedimensional folding, become sufficiently close to the free cysteine(e.g. cys²²³) of the hinge region to form a non-natural disulfide bondthat may alter the structure and biological activity of the fused targetprotein. The inventors' hypothesis may partially explain why there hasyet to be any clinically-proven success in attempts to create functionalfusion proteins with widely-used growth factors such as EPO, G-CSF andGM-CSF, etc.

Previous reports have used various methods to create fusion proteinsbetween a therapeutic protein and an Fc fragment/immunoglobulinmolecule. In most of these reports, researchers changed amino acidsequences of the target protein, added a linker peptide between theC-terminal of the target protein and the N-terminal of the hinge regionof Fc fragment, or truncated the hinge region of the Fc fragment of thehinge region (resulting in the removal of the free cysteine (e.g.cys²²³)).

In U.S. Pat. No. 5,908,626, a fusion protein of IFN β with a humanimmunoglobulin Fc fragment is described which was linked by a syntheticoligopeptide (GGS)₂(GGGS)₂ [6]. The inventors in that patent believethis linker can “reduce the possibility of generating a new immunogenicepitope (a neoantigen) at what would otherwise be the fusion point ofthe IFN β and the immunoglobulin Fc fragment”. In U.S. Pat. Nos.6,797,493, 6,900,292, 7,030,226, 7,226,759, and 7,232,668, the hingeregion was replaced by a 16-amino acid peptide linker GS(GGGS)₃GS [10,12, 13, 20, 21]. In addition to the genetic approach, chemicalmanipulation has also been used to address the problem of non-nativedisulfide bonds. For example, the inventors in U.S. Pat. No. 6,808,902developed a process for treating an IL-1ra-Fc fusion protein with acopper (II) halide in order to prevent or correct a non-native disulfidebond which caused misfolding of that fusion protein [12]. An Fc-EPOfusion protein (rather than the conventional EPO-Fc fusion) has shownpoor pharmacokinetics and little EPO efficacy in mice; mutation of fouramino acids of the EPO molecule is required to obtain a functionalFc-EPO fusion protein [30].

As mentioned above, the hinge region plays the role of the flexiblejunction region between the fused therapeutic protein and the Fcfragment (CH2 and CH3). Truncation or significant changes of the hingeregion may have undesirable effects on ability of the hinge region toact as flexible junction. The addition of peptide linkers may not onlyimpair the natural conformation of the fusion protein but also greatlyincrease the risk of immunogenecity by introducing a non-nativestructure.

The need exists for therapeutic protein/Fc fragment fusion proteins thathave a prolonged half-life and/or enhanced activity without increasingthe risk of an immunogenic response.

SUMMARY OF THE INVENTION

According to one aspect of the present invention, a fusion proteinhaving a non-immunoglobulin polypeptide having a cysteine residueproximal to the C terminal thereof, and an immunoglobulin component witha mutated hinge region is provided. The mutation comprises a pointmutated site corresponding in position to the position in a native hingeregion of the cysteine residue located nearest the cysteine residue ofthe non-Ig component. The distance from the cysteine residue of thenon-immunoglobulin polypeptide and any remaining cysteine residues ofthe mutated hinge region is sufficient to prevent the formation of adisulphide bond therebetween.

According to one aspect of the present invention a fusion protein havinga non-immunoglobulin polypeptide and an immunoglobulin component isprovided. The immunoglobulin component has a mutated hinge region. Themutation comprises a point mutated site in a hinge region of theimmunoglobulin component promixate to the non-immunoglobulinpolypeptide. A cysteine residue of the hinge region is substituted by anon-cysteine residue.

According to one aspect of the present invention, a fusion proteinhaving a non-immunoglobulin polypeptide directly linked to a humanimmunoglobulin component is provided. The fusion protein has a prolongedhalf-life in vivo in comparison to naturally occurring or recombinantnative non-immunoglobulin polypeptide.

According to one aspect of the invention, multimeric proteins comprisinga plurality of the fusion proteins according to the foregoing aspects ofthe invention are provided.

According to one aspect of the invention, methods of producing fusionproteins according to the foregoing aspects of the invention areprovided. The methods include the step of culturing a cell linetransfected with a DNA molecule that encodes the sequence of the fusionprotein and purifying the encoded protein.

According to one aspect of the invention, methods of stimulating whiteblood cell production in a mammal are provided, wherein the methodsinclude the step of administering to the mammal a fusion proteinaccording to the foregoing aspects of the invention.

According to one aspect of the invention, pharmaceutical compositionsincluding a fusion protein according to the foregoing aspects of theinvention and a pharmaceutically acceptable carrier, adjuvant or diluentare provided.

According to one aspect of the invention, methods of stimulating whiteblood cell production in a mammal are provided, wherein the methodsinclude the step of administering to the mammal a pharmaceuticalcomposition according to foregoing aspects of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

In drawings intended to illustrate various embodiments of the inventionbut which are not intended to be constructed in a limiting manner.

FIGS. 1A to 1C are schematic diagrams illustrating the strategy forgenerating point mutations and the amplification of the mutated wholegene encoding a Fc fusion protein simultaneously by overlapping PCR. A:A base-pair mutation was introduced into primers P2 and P3. Theoverlapping fragments of non-Ig and Fc with the desired mutation wereamplified by p1/p3 and p2/p4 respectively. B: The mixed fragments from Aserved as a template. The whole molecule with the desired mutation wasamplified by p1/p4. C: The resulting whole molecule with the desiredmutation. X represents the non-Ig moiety of fusion protein.

FIG. 2A is a schematic diagram showing the general structure of the DNAmolecule encoding the recombinant human EPO-FcG fusion protein(rHuEPO-FcG) in which the first cysteine from the N-terminal of thehinge region (cys²²³) is substituted by glycine and used as the mutanthinge region for the construction of an EPO-Fc fusion protein. Thismutant hinge region-containing Fc fragment is referred to as FcG, andthe Fc fragment containing the hinge region with the native cysteine atthe sixth residue from its N-terminal as FcC respectively.

FIG. 2B is a sequence listing showing the nucleotide sequence [SEQ IDNO:1] and the deduced amino acid (aa) sequence [SEQ ID NO:2] ofrHuEPO-FcG protein. The total length of DNA is 1281 bp. The 426 aminoacids in the deduced protein sequence include 27 aa for the signalpeptide and 399 aa for the complete rHuEPO-FcG protein. The completerHuEPO-FcG protein consists of human EPO domain (166 aa), hinge region(16 aa, underlined), and CH2 and CH3 domains (217 aa) of the Fc fragmentof human IgG1. The calculated molecular weight of the polypeptide of themature rHuRPO-FcG fusion protein is 44.6 kDa, composed of 18.5 kDa(41.4%) of EPO fragment and 26.1 kDa (58.6%) of IgG 1 Fc fragment. Ahomodimer is formed by two disulfide bonds via the two cysteine residues(boxed) within the hinge region. At residue 172 of the mature fusionprotein (i.e. the 6^(th) amino acid of hinge region) the native cysteineresidue has been substituted by glycine (bold).

FIG. 3A is a schematic diagram showing the general structure of the DNAmolecule encoding the wild type human EPO-FcC fusion protein(rHuEPO-FcC) in which the first cysteine from the N-terminal of thehinge region (cys²²³) is maintained.

FIG. 3B is a sequence listing showing the nucleotide sequence [SEQ IDNO:3] and the deduced amino acid (aa) sequence [SEQ ID NO:4] of a wildtype rHuEPO-FcC protein. The sequence particulars are the same as shownin FIG. 2B except that the native, wild type cysteine residue ismaintained at residue 172 of the mature fusion protein (i.e. the 6^(th)amino acid of the hinge region).

FIG. 4A is a schematic diagram showing the general structure of the DNAmolecule encoding the fusion protein between native GM-CSF molecule andthe FcG fragment (HuGMCSF-FcG).

FIG. 4B is a sequence listing showing the nucleotide sequence [SEQ IDNO:5] and the deduced amino acid (aa) sequence [SEQ ID NO:6] ofrHuGMCSF-FcG fusion protein. The total length of DNA is 1131 bp. The 377amino acids in the deduced protein sequence include 17 aa for the signalpeptide and 360 aa for the complete HuGMCSF-FcG fusion protein. Thecomplete rHuGMCSF-FcG fusion protein consists of complete GM-CSFmolecule (127 aa), mutant hinge fragment (16 aa, underlined), and CH2and CH3 domains (217 aa) of the Fc fragment of human IgG 1. Thecalculated molecular weight of mature rHuGMCSF-FcG fusion protein is40.6 kDa, composed of 14.5 kDa (35.7%) of GM-CSF fragment and 26.1 kDa(64.3%) of IgG 1 Fc fragment. A homodimer is formed by two disulfidebonds via the two cysteine residues (boxed) within the hinge region. Atresidue 150 of the fusion protein (i.e. the 6^(th) amino acid of hingeregion) the native cysteine residue has been substituted by glycine(bold).

FIG. 5 is an image showing the sizes of the dimeric form of purerHuEPO-FcG protein in non-reduced condition and monomeric form of purerHuEPO-FcG protein in reduced condition by SDS-PAGE analysis. Thepurified rHuEPO-Fc protein from the supernatants of the cultured CHOcell-line expressing rHuEPO-FcG exists mainly as the dimeric form andhas a molecular weight of about 180 kDa on 8% bis-tris gel innon-reduced condition. In reduced condition (100 mM dithiothreitol(DTT)) to break disulfide bonds, the dimer is separated into twoidentical monomeric units with a molecular weight of 75 kDa.

FIGS. 6A and 6B are graphs showing the dose-dependent increase ofhemoglobin (Hb) levels in normal mice treated three times per week witha subcutaneous injection (s.c.) of rHuEPO-FcG or rHuEPO. Each pointrepresents the mean Hb level of the group (6 mice). Day 0 levelsrepresent the Hb levels before treatment. A: Mice treated withrHuEPO-FcG. B: Mice treated with native rHuEPO.

FIGS. 7A and 7B are graphs showing the dose-dependent increase ofhemoglobin (Hb) levels in normal mice treated with once per week s.c. ofrHuEPO-FcG or rHuEPO. Each point represents the mean Hb level of thegroup (6 mice). Day 0 levels represent the Hb levels before treatment.A: Mice treated with rHuEPO-FcG. B: Mice treated with native rHuEPO.

FIGS. 8A and 8B are graphs showing the increase of hemoglobin (Hb)levels in normal mice treated with intravenously injection (i.v.) of12.5 μg/kg of rHuEPO-FcG or rHuEPO. Each point represents the mean Hblevel of the group (6 mice). Day 0 levels represent the Hb levels beforetreatment. A: Mice with treatment once a week. B: Mice with treatment 3times a week.

FIG. 9 is a graph showing the dose-dependent increase of hemoglobin (Hb)levels in 5/6 nephrectomized rats treated with once per week s.c. ofrHuEPO-FcG, rHuEPO or darbepoetin-alfa (abbreviated Darbe.). Each pointrepresents the mean Hb level of the group. Normal controls were normalrats with injection of carrier solution. Model controls were the 5/6nephrectomized rats with injection of carrier solution. Week 0 levelsrepresent the Hb levels before treatment. *: week(s) post treatment.

FIG. 10 is a graph showing the dose-dependent increase of hemoglobin(Hb) levels in 5/6 nephrectomized rats treated once every two weeks s.c.with rHuEPO-FcG, rHuEPO or darbepoetin-alfa (abbreviated Darbe.). Eachpoint represents the mean Hb level of the group. Normal controls werenormal rats with injection of carrier solution. Model controls were the5/6 nephrectomized rats with injection of carrier solution. Week 0levels represent the Hb levels before treatment. *: week(s) posttreatment.

FIG. 11 is a graph showing the dose-dependent increase of hemoglobin(Hb) levels in 5/6 nephrectomized rats treated once every two weeks i.v.with 62.5 μg/kg of rHuEPO-FcG, or darbepoetin-alfa (abbreviated Darbe.).Each point represents the mean Hb level of the group. Normal controlswere normal rats with injection of carrier solution. Model controls werethe 5/6 nephrectomized rats with injection of carrier solution. Week 0levels represent the Hb levels before treatment. *: week(s) posttreatment.

FIGS. 12A to 12C are graphs comparing the potency of rHuEPO-FcG, rHuEPOand darbepoetin-alfa in stimulating the colony formation of CFU-E andBFU-E in 5/6 nephrectomized rats treated with different doses andschedules. rHuEPO-FcG and darbepoietin-alpha (abbreviated Darbe.)treatment showed similar dose-dependent potencies for stimulating theCFU-E and BFU-E colony formation, while rHuEPO was less potent. A: s.c.once every week. B: s.c. once every 2 weeks. C: i.v. once every twoweeks.

FIG. 13 is a graph showing the serum levels of rHuEPO-FcG and rHuEPOafter the intravenous injection of 5 μg/kg of rHuEPO-FcG or rHuEPO toRhesus monkeys (mean levels of 5 monkeys).

FIG. 14 is a graph showing the dose-dependent increase of hemoglobin(Hb) levels in normal mice treated three times per week withsubcutaneous injection (s.c.) of rHuEPO-FcG, rHuEPO-FcC and rHuEPO. Eachpoint represents the mean Hb level of the group (8). Normal control wasnormal mice with injection of carrier solution. Day 0 levels representthe Hb levels before treatment.

FIG. 15 is a graph showing the dose-dependent increase of hemoglobin(Hb) levels in normal mice treated three times per week withsubcutaneous injection (s.c.) of rHuEPO-FcG, rHuEPO-FcC and rHuEPO. Eachpoint represents the mean Hb level of the group (8). Normal control wasnormal mice with injection of carrier solution. Day 0 levels representthe Hb levels before treatment.

FIG. 16 is a graph comparing the growth of white blood cells (WBC) indogs with experimental neutropenia by rHuGMCSF-FcG or by rHuGMCSF. ⁶⁰Coγ-ray irradiated dogs were treated s.c. with 10 μg/kg of rHuGMCSF-FcGevery other day, 20 μg/kg of rHuGMCSF-FcG every other day, or 20 μg/kgof rHuGMCSF every day. Day −1 and day 1 represent the day beforeirradiation and the next day after irradiation respectively. Treatmentstarted from day 1 and lasted for 10 days. Clinical observation andexamination of WBC counts lasted for 28 days. Each point represents themean WBC counts of the group. Model controls were the irradiated dogswith injection of carrier solution only.

DETAILED DESCRIPTION OF THE INVENTION

Throughout the following description specific details are set forth inorder to provide a more thorough understanding of the invention.However, the invention may be practiced without these particulars. Inother instances, well known elements have not been shown or described indetail to avoid unnecessarily obscuring the present invention.Accordingly, the specification and drawings are to be regarded in anillustrative, rather than a restrictive sense.

This invention relates to recombinant human fusion proteins combining atarget protein or polypeptide, such as a non-immunoglobulin polypeptide,with a Fc fragment or immunoglobulin molecule. The Fc fragment or Igmolecule includes a mutant hinge region wherein the first cysteine fromthe N-terminal of the hinge region (e.g. in human IgG 1, the sixth aminoacid, cys²²³) is replaced by a non-cysteine residue, such as anon-charged, non-polar amino acid (neutral amino acid). The result is amutant hinge region which maintains the natural length and flexibilityof the hinge region without the free cysteine that may lead to theformation of a non-natural disulfide bond with a cysteine in thenon-immunoglobulin polypeptide. The mutant hinge region may comprise thewhole or part of the human Fc fragment, or the whole or part of a humanimmunoglobulin molecule.

When any non-immunoglobulin polypeptide is directly fused to the hingeregion of the Fc fragment or Ig molecule to form a protein-Fc orprotein-Ig fusion protein, respectively, it is believed the mutant hingeregion lacking the N-terminal free cysteine allows the fused targetpolypeptide to maintain its structure, folding and biological functions.In particular, it is believed that any cysteine residues proximal to theC terminal of the target non-immunoglobulin polypeptide are thusprevented from forming any unexpected disulphide bonds with theN-terminal free cysteine found in the native hinge region. Cysteineresidues “proximal” to the C terminal of the non-immunoglobulinpolypeptide include those proximal with reference to position along theamino acid chain and as well to those proximal as result ofthree-dimensional folding of the non-immunoglobulin polypeptide. Thefusion proteins created with this mutant hinge region have near 100%sequence identity to natural sequences of both the target protein andhinge region, and therefore possess minimum immunogenecity risks.

The first cysteine from the N-terminal of the hinge region of human Fcfragment/immunoglobulin (e.g. the sixth amino acid, cys²²³, in humanIgG 1) may be substituted with any non-charged, non-polar amino acid(neutral amino acid). In the example disclosed herein, glycine was usedto replace the free cysteine. A person skilled in the art wouldappreciate that other amino acids could also replace the free cysteine.

The mutant hinge region that is formed by substituting the free cysteinenear its N-terminal as part of the Fc fragment or Ig molecule provides amethod or platform for generically producing fusion proteins between anynon-immunoglobulin polypeptide and Fc/Ig to prevent non-naturallyoccurring disulfide bonds, and thus retain the biological functions ofthe fused target polypeptide.

Several methods can be used to make the desired point mutation. Onemethod, described in Example 1 of this invention, adopts overlapping PCRto amplify the whole nucleic acid sequence of the fusion protein. Withspecifically-designed oligo primers, the desired point-mutation can beintroduced into the resulting nucleic acid sequence following geneamplification. Other methods, such as the Quick-Change™ mutagenesismethod from Invitrogen or artificial gene synthesis can also be used toproduce the point mutation. A person skilled in the art would appreciatethat any number of different methods could be used to substitute thefree cysteine of the hinge region (e.g. cys²²³).

The fusion protein created by using the mutant hinge region according toan embodiment of the present invention comprises a non-Ig moiety linkedto an Fc fragment of IgG expressed by the formula “X-hingeregion-CH2-CH3”, wherein X represents the non-Ig moiety, and CH2 and CH3represent two heavy chain domains of the Fc fragment of IgG.

The hinge region refers to the region between the CH1 and CH2 heavychain domains that contains the interchain disulfide bonds. Flexibilityin this region allows the molecules on both sides of hinge region tomove independently. The heavy chains are also glycosylated in thisregion, which helps protect this relatively exposed area againstdegradation. In one embodiment, the non-Ig moiety of the fusion proteinlinks to the hinge region directly, i.e. the C-terminal of the non-Igmoiety is directly fused to the N-terminal of hinge region. The firstcysteine (e.g. cys²²³) from the N-terminal of the hinge region may besubstituted by a non-charged, non-polar amino acid, such as glycine. Insome embodiments, a synthetic linker, such as (G4S)3 or G4SG5S, may beinserted between the non-Ig moiety and Fc fragment to ensure each partfolds properly. In other embodiments, one or more of the amino acidresidues upstream of the first cysteine (e.g. cys²²³) site may beremoved.

The non-immunoglobulin polypeptide may be, but is not limited to, anypeptide or polypeptide sequence with human or non-human origin, havingcomplete or non-complete amino acid sequences corresponding to anydefined human and non-human proteins, exhibiting biological functions ornon-biological functions, made artificially or obtained naturally. Thenon-immunoglobulin polypeptide may also be a variant of any proteinsdefined or non-defined before. These variants include but are notlimited to polypeptide sequences modified from a native protein sequencebut still partially or completely retaining its biological functions.Modifications include but are not limited to substitution, addition,insertion, deletion, or rearrangement of the amino acids of the nativepolypeptide sequences.

The non-immunoglobulin polypeptide may, for example, be a cytokine.“Cytokine” is used herein to describe proteins, analogs thereof, andfragments thereof which are produced by and excreted from a cell, andwhich elicit the biological response by binding with correspondingreceptors. Cytokines include but are not limited to hematopoieticfactors such as EPO, GM-CSF and granulocyte colony stimulating factor(G-CSF), interferons such as IFN α, IFN γ and IFN γ, interleukins suchas IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-11, IL-13, IL-14, IL-15,IL-16 and IL-18, tumor necrosis factors such as TNF α, and lymphokinessuch as lymphotoxin.

The non-immunoglobulin polypeptide may also be a ligand-binding proteinthat may block a receptor-ligand interaction at the cell surface, orneutralize the biological activity of another molecule in the bodyfluids. Ligand-binding proteins include but are not limited to CDsmolecules, CTLA-4, TNF receptors, and interleukin receptors.

The non-immunoglobulin polypeptide may also be a hormone, aneurotrophin, a neutrophin receptor (e.g. Trk A), a body-weightregulator, a serum protein, a clotting factor, a protease, anextracellular matrix component, an angiogenic factor, an anti-angiogenicfactor, an immunoglobulin receptor (e.g. IgG receptor), a blood factor(e.g. Factor VIII, Factor IX, Factor X), a cancer antigen (e.g. PSA,PSMA), a statin (e.g. endostatin, angiostatin) a therapeutic peptide ora growth-factor (e.g. Flt-3).

The non-immunoglobulin polypeptide may also be a non-human ornon-mammalian protein, or even a protein toxin. Examples include gp120,HIV transactivators, surface proteins from other viruses such as HBV,HCV and RSV, and parasitic surface proteins such as malarial antigens.

A recombinant vector with the nucleic acid sequence encoding the Fcfragment containing a mutant hinge region may be constructed. Thisvector, which possesses all the elements needed for its propagation,selection, and screening in either prokaryotic cells (such as E. coli)or eukaryotic cells (such as CHO cells), can serve as a platform toexpress Fc fusion proteins described in this invention. By using thisplatform, the nucleic acid sequence encoding the non-Ig moiety of thefusion protein is conveniently inserted in-frame into the vector at the5′-end of nucleic acid sequence encoding the mutated Fc moiety bymolecular cloning techniques.

As a specific example, a novel fusion protein having enhancederythropoietic properties was produced according to the presentinvention. The fusion protein, referred to herein as rHuEPO-FcG,comprises a human EPO molecule genetically linked to an immunoglobulinFc fragment containing a mutant hinge region of the present invention.The nucleic acid sequence of the rHuEPO-FcG fusion protein of thepresent invention is shown in SEQ ID No: 1, and the correspondingdeduced amino acid sequence is shown in SEQ ID No: 2. As discussedfurther below, the fusion protein may be in the form of a dimercomprising two identical polypeptide subunits. Each polypeptide subunit,from the N-terminal to C-terminal, includes the polypeptide sequence ofthe human EPO molecule, and the polypeptide sequence of the hingeregion, CH2 domain and CH3 domains of the Fc fragment of humanimmunoglobulin IgG 1. The two polypeptide subunits are joined bydisulfide bonds between the respective hinge regions to form the dimerstructure. The dimer has the same general shape as an IgG molecule andexhibits better stability than free EPO molecules as demonstrated in theexamples below.

As will be apparent to a person skilled in the art, the hinge region ofan intact immunoglobulin provides the protein sufficient flexibility foreffective antigen-antibody binding. Similarly, in the present invention,the hinge region in which the free cysteine (e.g. cys²²³) is substitutedby glycine is included in the design of the rHuEPO-FcG fusion protein tomaintain its flexibility, particularly when the fusion protein is in thedimer form. As described below, this likely allows the normal binding ofthe EPO portion of the rHuEPO-FcG fusion protein to EPO receptors toeffect the biological functions of EPO. It is believed that the dimerform of the rHuEPO-FcG fusion protein, by providing two EPO molecules,is capable of inducing optimal activation of EPO receptors (for example,by facilitating receptor cross-linking).

As demonstrated in the examples set forth below, the rHuEPO-FcG fusionprotein has been successfully synthesized using recombinant DNAtechniques. The fusion protein has been shown in mice, rat and primatestudies to exhibit a prolonged in vivo half-life and enhancederythropoietic properties in comparison to naturally occurring orrecombinant native human EPO. The rHuEPO-FcG fusion protein containingthe mutant hinge region exhibits normal or even enhanced erythropoieticfunctions in normal animals and animals with experimental anemia. Thehalf-life of this fusion protein in circulation in primate studiesreached 37 hours in comparison to 8 hours for native humanerythropoietin and 24 hours for ARANESP from Amgen. As used in thispatent application, the terms “native human erythropoietin” and “nativehuman EPO” mean EPO having an identical and complete amino acid sequenceof the wild type EPO molecule. As will be appreciated by a personskilled in the art, native human EPO may be naturally occurring orrecombinantly produced (e.g. rHuEPO alpha). The term “native human EPO”does not include rHuEPO analogs, such as darbepoetin alpha where the EPOstructure has been significantly modified, such as byhyperglycosylation.

The nucleic acid sequence of the rHuEPO-FcG fusion protein of thepresent invention is shown in FIG. 2B. The complete rHuEPO-FcG fusionprotein is 399 amino acids in length. As shown in FIG. 2B, the completerHuEPO-FcG fusion protein consists of the EPO domain (166 amino acids),the hinge region (16 amino acids, underlined) and the CH2 and CH3domains (217 amino acids). A signal or leader peptide sequenceconsisting of 27 amino acids is also shown in FIG. 1B. The signalpeptide is cleaved during synthesis of rHuEPO-FcG.

As shown best in FIG. 2B, the EPO domain has a cysteine residue near itsC-terminal (amino acid number 161). The mutant hinge region includes 2cysteine residues, at amino acid numbers 178 and 181 which are boxed inFIG. 2B. The hinge region cysteine residues form the disulphide bondsbetween the polypeptide subunits of the homodimer as discussed above.The naturally occurring hinge region of a human IgG 1 fragment also hasa cysteine at residue number 6 of the hinge region portion (measuredfrom the N-terminal). According to an embodiment of the presentinvention, the cysteine residue 6 of the hinge portion has beensubstituted by a non-cysteine residue. In particular, in the embodimentof FIG. 2B, the cysteine has been substituted by glycine (at amino acidresidue 172 of rHuEPO-FcG, which corresponds to residue 6 of the hingeregion). As will be apparent to a person skilled in the art, othernon-cysteine residues could also be substituted for cysteine at thislocation to avoid formation of a disulfide bond.

As a result of the amino acid substitution at residue 172, the firstcysteine residue of the hinge region (at residue 178) is spaced 17 aminoacids from the above-described cysteine residue of the EPO domain (atresidue 161). The inventors believe that the minimum spacing between thecysteine residue 161 of the EPO domain and the first cysteine residue ofthe hinge region should be at least 12 amino acids to enable successfulassembly and/or EPO receptor binding of a homodimer of rHuEPO-FcG. Thatis, if residue 172 is a cysteine residue, an undesirable disulfide bondmay potentially be formed, such as between cysteine residues 161 and172. This may alter the three dimensional structure of the EPO molecule,resulting in the impairment and/or the loss of the biological functionsof EPO.

In one embodiment of the invention, the EPO domain is linked directly tothe Fc fragment portion of the fusion protein. By avoiding an externallinker peptide, the preferred three dimensional structure of therHuEPO-FcG fusion protein is maintained and the risk of triggering anundesirable immunogenic response is minimized. The hinge region of theFc fragment is preferably at least 9 amino acids in length and ispreferably in the range of about 10 to 20 amino acids in length.

As another specific example, a fusion protein combining human GM-CSF andthe mutated Fc fragment was also produced genetically (rHuGMCSF-FcG).The nucleic acid sequence of the rHuGMCSF-FcG fusion protein of thepresent invention is shown in SEQ ID No: 5. The corresponding deducedamino acid sequence is shown in SEQ ID No: 6. In vivo experiments inanimals with experimental neutropenia demonstrate that rHuGMCSF-FcGexhibits enhanced biological functions in twills of stimulating thegrowth of white blood cells (WBC) as compared to rHuGMCSF.

Accordingly, two fusion proteins formed by direct linking of the targetproteins, human EPO and GM-CSF, to the mutant hinge region in which thefree cysteine (e.g. the sixth amino acid, cys²²³, in human IgG 1) wassubstituted by glycine, showed at least full biological functions of thefused target proteins, as compared to their natural molecules. Theseresults strongly suggest that the mutant hinge region of the presentinvention allows the direct fusion of a target protein to an Fcfragment/Ig molecule while retaining biological functions of the fusedtarget protein. The resulting fusion protein of target protein-Fc/Igcomplex, in addition to retaining natural biologic functions, exhibitssignificantly prolonged half-life in vivo.

In a further embodiment, the mutant hinge region without the freecysteine (e.g. the sixth amino acid, cys²²³, in human IgG 1) could beused as a standard platform for generating fusion proteins that havelonger circulation half-life and/or exhibit full or enhanced biologicalfunctions compared to the target protein. Unlike PEGylation, anadvantage of making fusion proteins of the present invented is that thebiological activity of the target protein will not be impaired and thepotential immunogenic responses are minimized since the fusion proteinhas almost 100% sequence identity to natural human proteins (i.e., onlyone amino acid is different).

EXAMPLES

The following examples will further illustrate the invention in greaterdetail although it will be appreciated that the invention is not limitedto the specific examples.

1. Generation and Amplification of Mutated Nucleic Acid SequencesEncoding the Fusion Proteins rHuEPO-FcG and rHuGMCSF-FcG

Full length DNA molecules encoding the amino-acid sequence of thepolypeptide of rHuEPO-FcG, rHuEPI-FcC (wild type) and rHuGMCSF-FcG weregenerated by overlapping PCR using the following oligo primers (QIAGENInc., US), respectively:

[SEQ ID NO: 7] EF5: 5′-ccggaattcgccaccatgggggtgcacgaatgtcctgcct-3′[SEQ ID NO: 8] EF3: 5′-ttttccttttgcggccgcttatttacccggagacagggagag-3′[SEQ ID NO: 9] EFL5: 5′-aggcctgcaggacaggggacagagttgagcccaaatctggtgaca-3′[SEQ ID NO: 10] EFL3:5′-tgtcaccagatttgggctcaactctgtcccctgtcctgcaggcct-3′ [SEQ ID NO: 11]EFL5w: 5′-aggcctgcaggacaggggacagagttgagcccaaatcttgtgaca-3′[SEQ ID NO: 12] EFL3w:5′-tgtcacaagatttgggctcaactctgtcccctgtcctgcaggcct-3′ [SEQ ID NO: 13] GF5:5′-actgaattcgccaccatgtggctgcagagcctgctgctcttgggcactgtggcctgc-3′[SEQ ID NO: 14] GF3: 5′-ttttccttttgcggccgcttatttacccggagacagggagag-3′[SEQ ID NO: 15] GFL5:5′-gactgctgggagccagtccaggaggttgagcccaaatctggtgacaaaac-3′ [SEQ ID NO: 16]GFL3: 5′-gttttgtcaccagatttgggctcaacctcctggactggctcccagcagtc-3′

An EcoR I site was introduced in EF5 and GF5, and a Not I site wasintroduced in EF3 and GF3. For optimal expression of the proteins inmammalian cells, the Kozak sequence (GCCACCATGG) was also introduced inEF5 and GF5. The pairs EFL5/EFL3 and EFL3w/EFL5w are complementarysequences consisting of the 3′-terminal DNA sequence of EPO (23 bp) andthe 5′-terminal DNA sequence of mutated and wild IgG 1 hinge (22 bp),respectively. The pair of GFL5/GFL3 are complementary sequencesconsisting of the 3′-terminal DNA sequence of GM-CSF (24 bp) and the5′-terminal DNA sequence of mutated IgG 1 hinge (26 bp).

A. Generation of Wild and Mutated Sequences of rHuEPO-FcG

First, an EPO DNA fragment of 0.6 kb was amplified by PCR (Platinum TaqDNA Polymerase High Fidelity) with primers EF5 and EFL3 or EFL3w fromplasmid p9E containing the full length of human EPO cDNA, mutated andwild Fc fragment of 0.7 kb with primers EF3/EFL5, and EF3/EFL5w fromplasmid pD containing the full length of human IgG1 cDNA sequence,respectively (p9E and pD are from the inventors' own lab). The twofragments were then purified and mixed in equal amount. Using the mix asa template, the full length mutated and wild rHuEPO-FcG DNA of 1.3 kbwas amplified by primers EF5 and EF3, respectively.

B. Generation of Mutated Sequence of rHuGMCSF-FcG

First, a GM-CSF DNA fragment of 0.4 kb was amplified by PCR (PlatinumTaq DNA Polymerase High Fidelity) with primers GF5 and GFL3 from plasmidpSS-GM containing the full length of human GMCSF cDNA, and a mutated Fcfragment of 0.7 kb was amplified with primers GF3/GFL5 from plasmid pDcontaining the full length of human IgG1 cDNA sequence (pSS-GM and pDare from the inventors' own lab). The two fragments were then purifiedand mixed in equal amount. Using the mix as a template, the full lengthof mutated rHuGMCSF-FcG DNA of 1.1 kb was amplified by primers GF5 andGF3.

2. Construction of the Recombinant Plasmids

Recombinant plasmids pCdEpo-FcG, pCdEpo-FcC and pCdGMCSF-FcG encodingthe fusion protein of rHuEPO-FcG, rHuEPO-FcC and rHuGMCSF-FcG,respectively, were constructed by cloning the amplified nucleic acidsequences from Example 1 into expression vector pCD1.

The purified three fragments were digested by EocR I and Not I (NewEngland Biolab Inc., USA) and then cloned into EcoR I/Not I-digestedmammalian expression vector pCD1 (FIG. 2). The resulting recombinantvectors were named pCdEpo-FcG, pCdEpo-FcC and pCdGMCSF-FcG. The insertednucleic acid sequences encoding the amino-acid sequence of therHuEPO-FcG, rHuEPO-FcC and rHuGMCSF-FcG were confirmed by DNAsequencing.

3. Establishment of rHuEPO-FcG, rHuEPO-FcC, and rHuGMCSF-FcG ExpressionCell Lines

Chinese hamster ovary (CHO) cells with dihydrofolate reductase (dhfr)deficiency (CHO/dhfr⁻, ATCC No. CRL-9096), which have been approved bythe FDA for biological substance production, were used as host cells forrecombinant expression of rHuEPO-FcG, rHuEPO-FcC, and rHuGMCSF-FcG.

The CHO-dhfr⁻ cells were transfected with the recombinant vectorspCdEpo-FcG, pCdEpo-FcC and pCdGMCSF-FcG using Lipofectamine (Gibco, Cat.No: 18292-037, USA). The supernatants from the culture of selectedclones were assayed by ELISA (Roche, Cat. No: 1-693 417 and Cat. No:HSGMO, Canada) for EPO and GM-CSF activity respectively. Positive cloneswere further screened under increasing methotrexate (MTX) pressures.Cell lines with highest protein expression were selected, and graduallyadapted to serum-free media (CD CHO Medium, Gibco, and Cat. No:10743-029, USA). These selected CHO cell lines were used for theproduction of rHuEPO-FcG, rHuEPO-FcC, and rHuGMCSF-FcG, respectively.

4. Purification of rHuEPO-FcG, rHuEPO-FcC, and rHuGMCSF-FcG Proteins

rHuEPO-FcG, rHuEPO-FcC, and rHuGMCSF-FcG protein molecules contained inthe supernatants collected from the serum-free media culturing theselected CHO cells were isolated at first by Protein A affinitychromatography (Amersham, Cat. No: 17-0402-01, Canada). The isolatedproteins were further purified by gel filtration in HiLoad 16/60Superdex 200 pg columns (Amersham, Cat. No: 17-1069-01, Canada). Thepurity of the rHuEPO-FcG, rHuEPO-FcC, and rHuGMCSF-FcG proteins was morethan 98% as determined by electrophoresis.

5. Determination of the Sizes of the Purified rHuEPO-FcG Protein

First, SDS-PAGE was carried out to determine the sizes of the purifiedrHuEPO-FcG, rHuEPO-FcC, and rHuGMCSF-FcG proteins. A single band withmolecular weight of about 180 kDa for rHuEPO-FcG and rHuEPO-FcC, and 140kDa for rHuGMCSF-FcG was seen on an 8% Bis-Tris gel under non-reducingconditions, which measures the overall size of the protein with theexistence of disulfide bonds. This indicated that most of theserecombinant protein molecules were produced in their dimeric form, asexpected from the design of the fusion protein. When SDS-PAGE analysiswas conducted under reducing conditions (100 mM dithiothreitol (DTT)) tobreak the disulfide bonds, only single bands with molecular weight of 75kDa and 60 kDa were identified, consistent with the estimated molecularweight of single polypeptide chains of HuEPO-hinge region-CH2-CH3 andHuGMCSF-hinge region-CH2-CH3, respectively.

The molecular weight of the purified rHuEPO-FcG fusion protein withglycosylation was determined by mass spectrometry (MALDI-TOF-MS) to be111099 daltons (111.1 kDa). In this assay, only a single peak of proteinwas observed, indicating that the purified rHuEPO-FcG protein was nearly100% pure. The 15 amino acids of the N-terminal of the pure rHuEPO-FcGprotein were determined by protein sequence analysis as:APPRLICDSRVLERY. This was consistent with the sequence of the first 15amino acids of the native human EPO polypeptide, and confirms that thepurified rHuEPO-FcG G protein does have the correct and complete EPOmolecule sequence as predicted by the DNA sequence encoding the aminoacid sequences of the rHuEPO-FcG G fusion protein.

6. Enhanced Erythropoietic Activities of rHuEPO-FcG in Normal Mice

In vivo experiments in mice were conducted to confirm the retention oferythropoietic activity of the rHuEPO-FcG protein and to determine itsefficacy compared to rHuEPO and darbepoetin-alpha. For comparisonpurposes, the doses of EPO used in the described animal experiments ofthe invention, namely rHuEPO-FcG, rHuEPO (i.e., native human EPO) anddarbepoetin-alpha, were compared according to the amount of EPO moleculeportion alone. In respect of rHuEPO-FcG protein, the EPO portioncontributes to 41.4% of the total rHuEPO-FcG molecular weight ascalculated by the ratio of the weight of amino acids of EPO to weight ofthe total amino acids of the whole rHuEPO-FcG molecule (166 of 399 aminoacids). The EPO amount for rHuEPO-FcG was thus determined to be 41.4% ofthe total amount of rHuEPO-FcG protein.

rHuEPO-FcG (stock concentration: 0.5 mg/ml, purity of 98.6%) and nativehuman rHuEPO (i.e. with natural human EPO structure) (6000 IU/0.5 ml,manufactured by Kirin Brewery Co., Japan) were diluted in carriersolution (2.5 mg/ml of human serum albumin, 5.8 mg/ml of sodium citrate,0.06 mg/ml of citric acid and 5.8 mg/ml of sodium chloride, pH 5.5-5.6).The dose of rHuEPO was calculated according to its activity/amountratio. BALB/c mice (6 to 8 weeks old, weighing 18-22 g, equal numbers ofmale and female, purchased from Experiment Animal Center, AMMS, China)were grouped randomly with 6 in each group. Each group of mice wastreated with one combination of one dose (0.1, 0.5, 2.5, 12.5, 62.5μg/kg), one injection route (i.v. through the tail vein or s.c.) and oneinjection schedule (three times per week or once per week). The controlgroup of mice was injected with an equal volume of carrier solution. Thetreatment lasted for 3 weeks and the total observation time was 5 weeks.Peripheral blood samples (tail vein) were taken before treatment, on the4^(th) day and 7^(th) day of every week for 5 weeks. Hemoglobin (Hb) wasmeasured as the index by absorptiometry. Mean±SD was calculated from thedata of each group and a t test was conducted among different groups.

The administration of EPO three times per week to mice, provided thatthe EPOs have normal erythropoietic activity, would induce saturatedstimulation of erythropoiesis. As shown in FIG. 4, both groups treated 3times per week s.c. had significant elevation of Hb levels even at thedose of 2.5 μg/kg. This experiment demonstrated that rHuEPO-FcGexhibited an in vivo erythropoietic activity as effective as rHuEPO. Theelevation of Hb levels in the treated group was dose-dependent. However,saturated elevation of the Hb levels was induced in mice at the dose of12.5 μg/kg of rHuEPO-FcG, whereas the similar saturated elevation of theHb levels was only achieved at the dose of 62.5 μg/kg of rHuEPO. Theelevation of Hb levels induced by 2.5 μg/kg of rHuEPO-FcG was alsogreater than that by 2.5 μg/kg of rHuEPO. These results suggest morepotent erythropoietic stimulation by rHuEPO-FcG compared to rHuEPO.

The erythropoietic potency of rHuEPO-FcG was further explored byreducing the injection times to once per week subcutaneously. As shownin FIG. 5, the rHuEPO-FcG-treated groups showed dose-dependent elevationof Hb levels at the doses of 12.5, or 62.5 μg/kg. Both doses of 12.5 and62.5 μg/kg of rHuEPO also induced the elevation of Hb levels to asimilar extent, which was much lower than that by 62.5 μg/kg ofrHuEPO-FcG. This strongly indicates that rHuEPO-FcG has enhancederythropoietic activity in vivo, presumably due to either the prolongedhalf-life of the rHuEPO-FcG in vivo or improved EPO receptorbinding/activation by the dimer EPO molecules in the rHuEPO-FcG protein,or by the combined effects of both.

When the same doses (12.5 μg/kg) of rHuEPO-FcG or rHuEPO wereadministrated intravenously either three times per week or once perweek, elevation of the Hb levels was observed for all the treated groups(FIG. 6). However, i.v. administration once per week of rHuEPO-FcGinduced greater, more persistent elevation of the Hb levels, whichcontinued longer after the treatment was over. This data providesfurther support for the enhanced erythropoietic properties of therHuEPO-FcG protein in comparison with rHuEPO having the structure ofnaturally occurring EPO protein.

7. Enhanced Erythropoietic Activities of rHuEPO-FcG in 5/6Nephrectomized Rats

Experiments in normal mice proved the enhanced erythropoietic activitiesof rHuEPO-FcG in vivo. To further observe the efficacy of rHuEPO-FcG instimulating erythropoiesis, pharmacodynamic studies were conducted inrats with experimental renal anemia induced by 5/6 nephrectomy. Theefficacy of rHuEPO-FcG was compared with those of rHuEPO anddarbepoetin-alpha (60 μg/ml, lot. No. N079, manufactured by KirinBrewery Co., Japan).

Wistar rats (male and female in equal number, weighing 160-180 g,purchased from Vitalriver Experiment Animal Inc., Beijing, China.Licence No. SCXK11-00-0008) were used to create the anaemia model due tothe renal functional failure by a two-step nephrectomy [27]. 5/6nephrectomy was performed on rats with general anaesthesia by twoseparate operations under sterile conditions. After ⅔ of the left kidneywas resected, the rats were allowed to recover for twenty days. Theright kidney was then resected. Antibiotics were administrated toprevent infection after each operation. In total, 5/6 of the kidneytissue from each rat was resected. The nephrectomized rats graduallydeveloped renal function insufficiency and anaemia. Anaemia stabilized50 days after the nephrectomy, and the rats were then randomly grouped(9 per group) to begin administration of the EPOs. Each group of ratswas treated with one combination of one dose (2.5, 12.5, 62.5 μg/kg),one injection route (i.v. through the tail vein or s.c.) and oneinjection schedule (once per week or once every 2 weeks). The controlgroup and model group of rats were injected with an equal volume ofcarrier solution. Treatment lasted for 4 weeks and the total observationtime was 6 weeks.

All doses (2.5, 12.5, 62.5 μg/kg) of rHuEPO-FcG, administratedsubcutaneously once per week, induced dose-dependent elevation of Hblevels compared to the model control group that did not receive EPOtreatment. Both 12.5 and 62.5 μg/kg of rHuEPO or darbepoetin,administrated subcutaneously once per week also induced elevation of Hblevels. The increased levels of Hb in both groups treated with 12.5 or62.5 μg/kg of rHuEPO-FcG were significantly higher than those in groupstreated with 12.5 or 62.5 μg/kg of rHuEPO respectively. The Hb levels in62.5 μg/kg of rHuEPO-FcG-treated group were also slightly higher thanthat in 62.5 μg/kg of darbepoetin-treated group. After stoppingtreatment, the decrease of Hb levels in the 62.5 μg/kg ofrHuEPO-FcG-treated group was much slower and the Hb levels remainedhigher than those of both normal control and model control groups untilthe end of observation (two weeks after treatment), indicating astronger and/or a prolonged erythopoietic stimulation (summarized inFIG. 7).

For treatment with subcutaneous injection once every two weeks, only12.5 or 62.5 μg/kg of the three EPOs were administrated (FIG. 8). 12.5μg/kg of rHuEPO barely increased Hb levels compared to the model controlgroup, and the weak erythropoietic response in the 62.5 μg/kg ofrHuEPO-treated group failed to bring the Hb levels to normal incomparison with the normal control group. Treatments of eitherrHuEPO-FcG or darbepoetin at the doses of 12.5 or 62.5 μg/kg inducedsignificant elevation of Hb levels that was higher than that of thenormal control group, indicating the effective correction of anaemia byboth rHuEPO-FcG and darbepoetin. No significant differences wereobserved between same doses of rHuEPO-FcG and darbepoetin in terms ofefficacy. The high dose of 62.5 μg/kg resulted in the persistentincrease of erythropoiesis until the termination of the observation (twoweeks post treatment). This further suggested that rHuEPO-FcG anddarbepoetin exhibit the property of long-lasting stimulation oferythropoiesis in vivo, which in turn could be translated to thereduction of administration frequency to patients clinically.

While darbepoetin has been approved for clinical application withless-frequent injections to increase patient compliance and reduce thework burden on health care providers, these experimental data stronglyindicate that rHuEPO-FcG disclosed herein has at least comparablepotential benefits. As discussed above, darbepoetin, as a mutant analogof the human EPO molecule containing additional sugar compounds(increased glycosylation), may have an increased risk of inducingimmunogenesis in vivo due to alteration of native three dimensionalstructures. Only long-term observation of patients undergoing treatmentwith darbepoetin will reveal the immunogenic risks of darbepoetin. Incontrast, rHuEPO-FcG, which does not modify the EPO molecule portion,has a carbohydrate content identical or closely similar to that ofnative human EPO. The amount of sialic acids in the inventors' purerHuEPO-FcG protein were approximately 10.0 mmol/mmol EPO, consistentwith the reported parameters of rHuEPO. The Fc portion of rHuEPO-FcG,with no external amino acid(s)/linking peptide, represents the generalstructure of human IgG 1, and should not generate an immunogenicresponse. If approved clinically, rHuEPO-FcG may provide a better choicefor patients than currently available rHuEPO and EPO analogs, especiallythose in need of long-term administration.

Intravenous injection once every two weeks, with each of rHuEPO-FcG anddarbepoetin (62.5 μg/kg), induced identical increases of Hb levels inthe rats with renal anaemia far above the normal Hb levels in the normalcontrol rats (FIG. 9). This further demonstrates the persistentstimulation of erythropoiesis by rHuEPO-FcG.

Data derived from cell culturing experiments of bone marrow cellscollected from the 5/6 nephrectomized rats after treatments (once perweek or per two weeks, s.c. or i.v.) showed that rHuEPO-FcG, rHuEPO anddarbepoetin all stimulated the formation of CFU-E and BFU-E. Thepotencies of rHuEPO-FcG and darbepoetin were similar and stronger thanthat of rHuEPO (FIG. 10).

Blood urinonitrogen (BUN) and creatinine levels were similar in thetreated groups and model control group. The levels of serum Fe in allthe treated groups were higher than that of the model control group.Pathological examinations revealed increased distribution of red bloodcell (RBC)-related cells in bone marrow and spleen of all EPO-treatedrats.

8. Pharmacokinetic Studies of rHuEPO-FcG in Rhesus Monkeys

As discussed above, the inventors have designed rHuEPO-FcG in such a waythat the EPO portion of the fusion protein retains the functionalproperties of natural EPO, such as stimulating erythropoiesis, and theFc fragment of human IgG 1 allows the retention of the fusion protein incirculation, thus extending its half-life in vivo. The above animalstudies have demonstrated the erythropoietic activities of rHuEPO-FcGare enhanced in comparison with rHuEPO. The inventors have alsoconducted pharmacokinetic studies to determine the in vivo half-life ofrHuEPO-FcG in comparison to that of rHuEPO. Primates were used togenerate data as they are biologically very similar to human beings.

Study design was based on literature reports and the experiments wereconducted according to the general guidelines of pharmacokinetics. Twogroups of Rhesus monkeys with 5 monkeys in each group (3-5 kg, purchasedfrom the Experiment Animal Center, AMMS, China) were injectedintravenously with 5 μg/kg of rHuEPO-FcG or rHuEPO, respectively. Bloodsamples were taken before and at 0.017, 0.167, 0.5, 1, 2, 4, 8, 12, 24,48, 96, 168 and 240 h after injection. Sera were collected bycentrifugation and the serum rHuEPO-FcG or rHuEPO levels were determinedby using human erythropoietin enzyme-linked immunosorbent assay (ELISA)kits (purchased from R&D Systems, Minneapolis, USA). The averagehalf-life of rHuEPO-FcG and rHuEPO injected intravenously was35.24+/−5.15 h and 8.72+/−1.69 h respectively.

To observe the bioavailability of rHuEPO-FcG, 5 μg/kg of rHuEPO-FcG wasinjected subcutaneously to 5 Rhesus monkeys. Blood samples were takenbefore and 1, 2, 5, 8, 10, 12, 15, 24, 48, 72, 96, 168 and 240 h afterthe injection, and the serum levels of rHuEPO-FcG were determined by theR&D kits. The bioavailability index was calculated as 35.71+/−5.37% withthe subcutaneous injection. This is essentially identical to thereported bioavailability figures of darbepoetin-alpha (ARANESP) inpatients with chronic renal failure.

This data demonstrates that rHuEPO-FcG has a significantly prolongedhalf-life in primates, and the in vivo half-life of rHuEPO-FcG is atleast four fold longer than that of rHuEPO manufactured by Kirin BeerBrewing Co. of Japan. The prolonged half-life in vivo likely contributesto the enhanced erythropoietic activity of rHuEPO-FcG.

9. Immunogenicity of rHuEPO-FcG in Macaca fascicularis

As indicated above, the design of rHuEPO-FcG fusion proteinintentionally avoids or minimizes changes of the immunogenic propertiesof the rHuEPO-FcG fusion protein. The inventors avoided including/addingany external amino acid(s) or linking peptide sequences in the fusionprotein. According to one embodiment of the invention, the HuEPO-Fcfusion protein shown in FIG. 1B only contains the polypeptide sequencesof the natural EPO protein and the Fc fragment (hinge region, CH2, CH3)of human IgG 1, and thus should not induce an immunogenic response northe production of antibodies against rHuEPO-FcG protein.

Primate studies were conducted to observe the immunogenicity ofrHuEPO-FcG protein. Ten crab-eating macaques (Macaca fascicularis)(male/female=5/5, ˜5 years old, average weight of male 4.0±0.3 kg,female is 2.9±0.4 kg, purchased from Laboratory Animal Center, AMMS,China) were injected subcutaneously with 5 μg/kg of purified rHuEPO-FcGthree times per week for four weeks, and two were injected with an equalvolume of carrier solution as the control animals. Sera were collectedonce a week for 5 weeks (1 week post-treatment) and tested for thespecific antibodies against rHuEPO-FcG by ELISA using purifiedrHuEPO-FcG (5 μg/ml) as the coating antigen. In addition, RBC counts andHb levels in the peripheral blood were also determined within theexperimental period. The resulting data shows that, while enhancement oferythropoiesis in the rHuEPO-FcG-treated macaques was observed (the meanRBC numbers increased from 4.74×10⁹/ml to 6.67×10⁹/ml and the mean Hblevels increased from 12.2 g/dl to 13.7 g/dl), rHuEPO-FcG did not inducethe production of any detectable specific antibodies against the fusionprotein. These results indicate that rHuEPO-FcG fusion protein does notcause immunogenicity in primates.

10. Acute Toxicity Studies of rHuEPO-FcG in Normal Mice

To assess the safety of rHuEPO-FcG fusion protein, acute toxic studieswere conducted in animals.

Two groups of BALB/c mice (n=20, equal numbers of male and female, 5-6weeks old, the average weight of female is 15.8+0.4 g, male is 15.9±0.6g, purchased from Chinese Academy of Medicine, China) were injectedintravenously once with an excessive amount of purified rHuEPO-FcG(male=13.3 mg/kg, female=13.2 mg/kg) or equal volume of the carriersolution via their tail veins. In addition to observing the instantreaction following injection, general behaviour and status, activities,eating and defecation patterns and changes were monitored and recordeddaily for 14 days. All mice were also weighed at day 7 and day 14. Atday 15 post-injection, anatomical examination of the main organs of themice were conducted. Pathologic examination were to be conducted if anyunusual changes or suspicious changes of the organs were observed.

All mice in the 2 groups had no obvious instant reaction followinginjection. Within the period of 14 days, no obvious changes ofbehaviour, activities, eating and defecation patterns were observed.Moreover, the weight of the mice in both groups increased steadilyduring the testing period, and no apparent differences were foundbetween the 2 groups on day 7 or day 14 post injection. No abnormal orpathologic changes were detected in the tissues of brain, lung, heart,liver and kidney. These results indicate that administration of anexcessive amount of rHuEPO-FcG, far more than required for exhibitingthe normal erythropoiesis function, is safe and had no apparent toxiceffects.

11. Comparison of Wild Type and Mutated Fusion Proteins of rHuEPO-FcCand rHuEPO-FcG

Investigations were also conducted to compare wild type and mutatedversions of proteins of HuEPO-Fc. As described above, in one embodimentthe invention includes a single amino acid mutation at amino acidresidue 172 (C172G).

In vivo experiments in mice were conducted to compare the erythropoieticactivity of the wild type fusion protein rHuEPO-FcG G with the mutatedfusion protein rHuEPO-FcG C and with recombinant human EPO (rHuEPO). Forcomparison purpose, all the doses of the three proteins used in thisexample, namely rHuEPO-FcG G, rHuEPO-FcG C and rHuEPO, were the amountsof the EPO molecule portion alone on a molar basis. In respect of therHuEPO-FcG G and rHuEPO-FcG C proteins, the EPO portion contributes41.4% of the total rHuEPO-FcG molecular weight as calculated by theratio of the weight of amino acids of EPO to the weight of the totalamino acids of the whole rHuEPO-FcG G and rHuEPO-FcG C molecules (i.e.,166 of 399 amino acids).

rHuEPO-FcG G (stock concentration: 300 μg/ml), rHuEPO-FcG C (stockconcentration: 90 μg/ml) and rHuEPO with the natural human EPO structure(6000 IU/0.5 ml, manufactured by Kirin Brewery Co., Japan) were dilutedin carrier solution (2.5 mg/ml of human serum albumin, 5.8 mg/ml ofsodium citrate, 0.06 mg/ml of citric acid and 5.8 mg/ml of sodiumchloride, pH 5.5-5.6). The dose of rHuEPO was calculated according toits activity/amount ratio. BALB/c mice (9 to 10 weeks old, weighing18-22 g, equal numbers of male and female, purchased from ExperimentAnimal Center, AMMS, China) were grouped randomly with 8 in each group.Each group of mice was treated with one combination of one dose (2.5,12.5, 62.5 μg/kg), one injection route (s.c.) and one injection schedule(three times per week or once per week). The control group of mice wasinjected with an equal volume of carrier solution. Treatment lasted for26 days. Peripheral blood samples (tail vein) for measurement were takenbefore treatment, on the 2^(nd), 6^(th), 9^(th), 13^(th), 16^(th),19^(th), 22^(nd) and 26^(th) days of treatment. Hb was measured as theindex by absorptiometry. Mean±SD was calculated from the data of eachgroup and a t test was conducted among different groups.

The administration of EPO three times per week to mice induced saturatedstimulation of erythropoiesis. As shown in FIG. 13, the mice treated byrHuEPO-FcG G 3 times per week s.c. had significant elevation of Hblevels even at the dose of 2.5 ug/kg at the 9^(th) day after treatment.The elevation of Hb levels in the treated group was dose-dependent.However, saturated elevation of the Hb levels was induced in mice at thedose of 12.5 ug/kg of rHuEPO-FcG G. The elevation of Hb levels inducedby 2.5 ug/kg of rHuEPO-FcG G was also greater than that by 2.5 ug/kg ofrHuEPO-FcG C and rHuEPO. These results suggested more potenterythropoietic stimulation by rHuEPO-FcG G.

The erythropoietic potency of rHuEPO-FcG G was further explored byreducing the injection times to once per week subcutaneously. As shownin FIG. 14, the rHuEPO-FcG G-treated groups showed dose-dependentelevation of Hb levels at the doses of 12.5, or 62.5 ug/kg. Both dosesof 12.5 and 62.5 ug/kg of rHuEPO also induced the elevation of Hb levelsto the similar extent, which was much lower than that by 62.5 ug/kg ofrHuEPO-FcG G. The rHuEPO-FcG C-treated groups showed significantly lowerHb levels. This strongly indicates that rHuEPO-FcG G has enhancederythropoietic activity in vivo. It is presumably due to either improvedEPO receptor binding/activation by the dimer EPO molecules in rHuEPO-FcGG protein or by the possible prolonged half-life of rHuEPO-FcG G invivo, or due to the combined effects of both.

The results demonstrated that rHuEPO-FcG G exhibited an enhanced in vivoerythropoietic activity compared to rHuEPO-FcG C and rHuEPO. Thisenhanced activity appears to be attributable to the single amino acidmutation in the hinge region of the recombinant molecule at residue 172(FIGS. 1 and 12). The results show that rHuEPO-FcG C exhibited verylittle erythropoietic activity in normal mice and much lesserythropoietic activity compared to rHuEPO-FcG G and rHuEPO.

12. Enhanced Therapeutic Effects of rHuGMCSF-FcG for Neutropenia in⁶⁰Co-Irradiated Dogs

The enhanced biological activity of rHuGMCSF-FcG was observed in modeldogs with neutropenia induced by ⁶⁰Co γ-ray irradiation. The efficacy ofrHuGMCSF-FcG was also compared with that of native GM-CSF.

rHuGMCSF-FcG (stock concentration: 1.8 mg/ml, purity 98%) and nativehuman rHuGM-CSF (150 μg/vial, manufactured by NCPC GeneTechBiotechnology Development Co., Ltd., China) were diluted in carriersolution (1% of HSA, 1.1% of benzyl alcohol, 40 mg/ml of mannitol, 10mg/ml of sucrose, and 1.2 mg/ml of tromethamine).

Beagle dogs (male and female in equal number, weighing 8-10 kg, 12-15months old, purchased from Beijing Xieerxin Institute of BioResources.Licence No. SOCK 2005-0005, China) were divided into 4 groups (4 in eachgroup) randomly: model control, low dose of rHuGMCSF-FcG (10 μg/kg),high dose of rHuGMCSF-FcG (20 μg/kg), and high dose of rHuGMCSF (20μg/kg). Each dog, with the pelvis shielded by lead blocks, wasirradiated by 6.5 Gy of ⁶⁰Co γ-ray at a rate of 295.54 rad/min. Thetreatment started from the next day of irradiation by subcutaneousinjection, once every other day for rHuGMCSF-FcG, or once every day forrHuGMCSF. The dogs from control group were injected with an equal volumeof carrier solution. Injections were given over 10 days and the totalobservation time was 28 days. After irradiation, the dogs were examinedeach day for their general clinical conditions. Every other day,peripheral blood was collected for examinations of WBC count, plateletcount, RBC count, HB, and hematocrit (Hct).

As shown in FIG. 16, all the irradiated dogs developed neutropeniaimmediately after irradiation. Since dogs from control group were onlyinjected with carrier solution, their clinical courses reflected naturalrecovery with no treatment. WBC counts decreased dramatically the dayfollowing irradiation and reached a nadir by day 4. The low WBC countlasted for another 4-5 days. After that, it began increasing slowly,indicating the recovery from neutropenia.

Dogs from the three administrated groups also developed similarneutropenia after irradiation, but their WBC counts showed rapid andsustained increases during the course of treatment. All the nadirs ofWBC counts were raised compared with control group. Furthermore, theduration of low WBC counts was also shorter. The increasing of WBCreached its peak on the day following the last injection. These resultsindicate that both rHuGMCSF-FcG and rHuGMCSF induced hematopoieticrecovery.

Different levels of activity were observed within the threeadministrated groups. rHuGMCSF-FcG demonstrated enhanced hematopoieticrecovery compared with rHuGMCSF. Despite only being administrated everyother day, the dogs injected with rHuGMCSF-FcG showed stronger recoverypotency than dogs with rHuGMCSF administrated daily. While the dogsinjected with low doses of rHuGMCSF-FcG (10 μg/kg) and high doses ofrHuGMCSF (20 μg/kg) showed similar WBC counts and recovery tendency, theWBC counts from dogs with high doses of rHuGMCSF-FcG (20 μg/kg) weretwice as high as the other two groups during the entire course ofrecovery. This dose-dependent, persistent elevation further indicatesthat rHuGMCSF-FcG possesses enhanced biological activity compared to itsnative counterpart (FIG. 16).

The enhanced biological activity of rHuGMCSF-FcG in vivo, similar tothat discussed in rHuEPO-FcG experiments, is also presumably due toimproved GM-CSF receptor binding/activation by the dimer GM-CSFmolecules in rHuGMCSF-FcG protein, the possible prolonged half-life ofrHuGMCSF-FcG in vivo, or due to the combined effects of both.

As will be apparent to those skilled in the art in the light of theforegoing disclosure, many alterations and modifications are possible inthe practice of this invention without departing from the spirit orscope thereof.

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1. A fusion protein comprising: a non-immunoglobulin polypeptidecomprising a cysteine residue proximal to the C terminal thereof; and animmunoglobulin component comprising a mutated hinge region, the mutationcomprising a point mutated site corresponding in position to theposition in a native hinge region of the cysteine residue locatednearest the cysteine residue of the non-Ig component, whereby thedistance from the cysteine residue of the non-immunoglobulin polypeptideand any remaining cysteine residues of the mutated hinge region issufficient to prevent the formation of a disulphide bond therebetween.2. A fusion protein according to claim 1, wherein the non-immunoglobulinpolypeptide is selected from the group consisting of a cytokine, aligand-binding protein, a hormone, a neurotrophin, a neutrophinreceptor, a body-weight regulator, a serum protein, a clotting factor, aprotease, an extracellular matrix component, an angiogenic factor, ananti-angiogenic factor, an immunoglobulin receptor, a blood factor, acancer antigen, a statin, a growth factor, a therapeutic peptide, anon-human protein, a non-mammalian protein and a protein toxin.
 3. Afusion protein according to claim 2, wherein the cytokine is selectedfrom the group consisting of hematopoietic factor, interferon,interleukin and tumor necrosis factor.
 4. A fusion protein according toclaim 3, wherein the hematopoietic factor is selected from the groupconsisting of erythropoietin, granulocyte-macrophage colony stimulatingfactor and granulocyte colony stimulating factor.
 5. A fusion proteinaccording to claim 2, wherein the ligand-binding protein is selectedfrom the group consisting of a CD molecule, CTLA-4, TNF receptor, andinterleukin receptor.
 6. A fusion protein according to claim 1, whereinthe human immunoglobulin component comprises an Fc fragment.
 7. A fusionprotein according to claim 6, wherein the Fc fragment is derived from anIgG.
 8. A fusion protein according to claim 7, wherein the IgG isselected from the group consisting of IgG 1, IgG 2, IgG 3 and IgG
 4. 9.A fusion protein according to claim 8, wherein the Fc fragment is a IgGFc fragment comprising the mutated hinge region and CH2 and CH3 domains.10. A fusion protein according to claim 9, wherein the IgG Fc fragmentderived from IgG
 1. 11. A fusion protein according to claim 1 whereinthe C-terminal of the non-immunoglobulin polypeptide is directly linkedto the N-terminal of the mutated hinge region.
 12. A fusion proteinaccording to claim 1, wherein the mutated hinge region comprises atleast 9 amino acids.
 13. A fusion protein according to claim 12, whereinthe mutated hinge region comprises between 10 and 20 amino acids.
 14. Afusion protein according to claim 1, wherein the point mutated sitecomprises a non-cysteine amino acid.
 15. A fusion protein according toclaim 1, wherein the point mutated site is the sixth amino acid positionmeasured from the N-terminal of the mutated hinge region and comprises anon-cysteine amino acid.
 16. A fusion protein according to claim 14,wherein the non-cysteine amino acid is a neutral amino acid.
 17. Afusion protein according to claim 14, wherein the non-cysteine aminoacid is glycine.
 18. A fusion protein according to claim 14, wherein thenon-cysteine amino acid is alanine.
 19. A fusion protein according toclaim 1, wherein the non-immunoglobulin polypeptide is a humangranulocyte-macrophage colony stimulating factor or a variant thereof.20. A multimeric protein comprising a plurality of fusion proteinsaccording to claim
 1. 21. A multimeric protein according to claim 20wherein the multimeric protein is a dimer.
 22. A method of producing afusion protein as defined in claim 1 comprising culturing a cell linetransfected with a DNA molecule that encodes the sequence of the fusionprotein and purifying the protein encoded thereby.
 23. A cell line asdefined in claim 22, wherein the cell line is a CHO cell line.
 24. Amethod of stimulating white blood cell production in a mammal comprisingadministering to the mammal a fusion protein according to claim
 19. 25.A method according to claim 24 wherein the mammal is a human.
 26. Apharmaceutical composition comprising a fusion protein according toclaim 19 and a pharmaceutically acceptable carrier, adjuvant or diluent.27. A method of stimulating white blood cell production in a mammalcomprising administering to the mammal a pharmaceutical compositionaccording to claim
 26. 28. A method according to claim 27 wherein themammal is a human.
 29. A fusion protein comprising: a non-immunoglobulinpolypeptide; and an immunoglobulin component comprising a mutated hingeregion, the mutation comprising a point mutated site in a hinge regionof said component promixate to said polypeptide, whereby a cysteineresidue of said hinge region is substituted by a non-cysteine residue.30. A fusion protein comprising a non-immunoglobulin polypeptide and ahuman immunoglobulin component, wherein the fusion protein has aprolonged half-life in vivo in comparison to naturally occurring orrecombinant native non-immunoglobulin polypeptide.
 31. A fusion proteinaccording to claim 30 wherein the non-immunoglobulin polypeptide isdirectly linked to the human immunoglobulin component.
 32. A fusionprotein according to claim 30 wherein the non-immunoglobulin polypeptideis linked to the human immunoglobulin component by a synthetic linker.33. A fusion protein according to claim 30, wherein the half-life of thefusion protein is at least three fold higher than the nativenon-immunoglobulin polypeptide.
 34. A fusion protein according to claim30, wherein the half-life of the fusion protein is at least four foldhigher than the native non-immunoglobulin polypeptide.
 35. A fusionprotein according to claim 30, wherein the fusion protein has enhancedbiological activity in comparison to the native non-immunoglobulinpolypeptide.
 36. A fusion protein according to claim 30, wherein thenon-immunoglobulin polypeptide is selected from the group consisting ofa cytokine, a ligand-binding protein, a hormone, a neurotrophin, aneutrophin receptor, a body-weight regulator, a serum protein, aclotting factor, a protease, an extracellular matrix component, anangiogenic factor, an anti-angiogenic factor, an immunoglobulinreceptor, a blood factor, a cancer antigen, a statin, a growth factor, atherapeutic peptide, a non-human protein, a non-mammalian protein and aprotein toxin.
 37. A fusion protein according to claim 36, wherein thecytokine is selected from the group consisting of hematopoietic factor,interferon, interleukin and tumor necrosis factor.
 38. A fusion proteinaccording to claim 37, wherein the hematopoietic factor is selected fromthe group consisting of erythropoietin, granulocyte-macrophage colonystimulating factor and granulocyte colony stimulating factor.
 39. Afusion protein according to claim 37, wherein the ligand-binding proteinis selected from the group consisting of a CD molecule, CTLA-4, TNFreceptor, and interleukin receptor.
 40. A fusion protein according toclaim 30, wherein the human immunoglobulin component comprises an Fcfragment.
 41. A fusion protein according to claim 40, wherein the Fcfragment is derived from an IgG.
 42. A fusion protein according to claim41, wherein the IgG is selected from the group consisting of IgG 1, IgG2, IgG 3 and IgG
 4. 43. A fusion protein according to claim 40, whereinthe Fc fragment comprises a hinge region and CH2 and CH3 domains.
 44. Afusion protein according to claim 43, wherein the hinge region comprisesat least 9 amino acids.
 45. A fusion protein according to claim 44,wherein the hinge region comprises between 10 and 20 amino acids.
 46. Afusion protein according to claim 45, wherein the hinge region ismutated.
 47. A fusion protein according to claim 46, wherein the hingeregion is point-mutated.
 48. A fusion protein according to claim 47,wherein the point-mutated site corresponds to the position of the firstcysteine from the N-terminal of a native hinge region.
 49. A fusionprotein according to claim 48, wherein the first cysteine is substitutedby a non-cysteine amino acid.
 50. A fusion protein according to claim49, wherein the non-cysteine amino acid is a neutral amino acid.
 51. Afusion protein according to claim 50, wherein the non-cysteine aminoacid is glycine.
 52. A fusion protein according to claim 50, wherein thenon-cysteine amino acid is alanine.
 53. A fusion protein according toclaim 30, wherein the non-immunoglobulin polypeptide is a humangranulocyte-macrophage colony stimulating factor or a variant thereof.54. A multimeric protein comprising a plurality of fusion proteinsaccording to claim
 30. 55. A multimeric protein according to claim 54wherein the multimeric protein is a dimer.
 56. A method of producing afusion protein as defined in claim 30 comprising culturing a cell linetransfected with a DNA molecule that encodes the sequence of the fusionprotein and purifying the protein encoded thereby.
 57. A cell line asdefined in claim 56, wherein the cell line is a CHO cell line.
 58. Amethod of stimulating white blood cell production in a mammal comprisingadministering to the mammal a fusion protein according to claim
 53. 59.A method according to claim 58 wherein the mammal is a human.
 60. Apharmaceutical composition comprising a fusion protein according toclaim 53 and a pharmaceutically acceptable carrier, adjuvant or diluent.61. A method of stimulating white blood cell production in a mammalcomprising administering to the mammal a pharmaceutical compositionaccording to claim
 60. 62. A method according to claim 61 wherein themammal is a human.
 63. A fusion protein comprising the amino acidsequence of SEQ ID NO:2 or a sequence substantially homologous thereto.64. A recombinant DNA molecule comprising the nucleic acid sequence ofSEQ ID NO:1 or a sequence substantially homologous thereto.
 65. A fusionprotein comprising the amino acid sequence of SEQ ID NO:6 or a sequencesubstantially homologous thereto.
 66. A recombinant DNA moleculecomprising the nucleic acid sequence of SEQ ID NO:5 or a sequencesubstantially homologous thereto.